| BackgroundCarbon disulfide (CS2) belongs to one of most important organic solvents and is widely used in industrial processes of fumigating grain, vulcanizing rubber, oil extraction, and viscose rayon fibers manufacturing. In the process of industrial production, CS2 is volatile and tends to spread to the production environment, which is harm to the exposed workers. Our country is a big textile country and the production of viscose fiber continues to grow rapidly. There are lots of large enterprises and millions of workers, including filament and staple fiber spinning process, and the main population is women employees.Some studies found that exposure to CS2 had toxic effects on the cardiovascular, neurological, liver, kidneys, endocrine system et al. In addition, the researches on reproductive toxicity of CS2 found that exposure to CS2 reproductive system had significant reproductive toxicity effects and it can lead to testicular atrophy, sexual dysfunction, the reduced count and the increased malformation rate of sperm in male workers, and gonadal dysfunction, abortion and birth defects of child generation in female workers. Our research group conducted a prospective study in CS2 exposure female workers and found that time to pregnancy was significantly longer in CS2 exposure female workers than that of the control, and the early pregnancy loss rate was significantly higher than the control, suggesting that CS2 could interfere with embryo implantation. With the animal studies, our group further found that exposure to CS2 at the peri-implantation resulted in the reduced number of implanted embryos, but its mechanism has not been elucidated yet. In recent years, studies of the signaling pathway in the reproductive process have become the concerned direction. mTOR (Mammalian target of rapamycin) was a kind of serine/threonine protein kinase, and is the core of cell growth, which was involved in gene transcription, protein translation, autophagy and a variety of biological activities. In addition, another study found that mTOR signaling pathway played an important role in the process of embryo implantation, and exogenous chemicals could interfere with the normal embryo implantation via the mTOR pathway. As the upstream regulatory factors of mTOR pathway, integrin β3 and LIF (leukemia inhibitory factor) were involved in the regulation of the expression of mTOR. Therefore, we assumed that after exposure to CS2, the abnormal expression of mTOR and its upstream regulators can lead to the disordered embryo implantation.In this study, we built an animal model in which pregnant mice were exposed to CS2 at the different times during the period of embryo implantation to detect the expression of mTOR, integrin P3 and LIF in uterine tissue, in order to explore the role of mTOR and related regulatory proteins in mechanism of embryo implantation disorders induced by CS2.ObjectiveTo built the animal model of embryo implantation disorders induced by CS2 exposure at different time points of embryo implantation in mice and observe the maternal toxicity and embryotoxicity by CS2 exposure at different time points and detect the expression levels of mTOR, integrin β3 and LIF in uterine tissue at different times of exposure, in order to explore the role of mTOR and related regulatory proteins (integrin β3 and LIF) in mechanism of embryo implantation disorders induced by CS2, further to reveal the mechanism of embryo implantation disorders induced by CS2 for providing a scientific basis of the prevention and control of female reproductive damage to CS2.MethodsStudy design:The study was divided to 3 parts:Part 1 was established to build a time-dependent animal mode after CS2 exposure during the period of embryo implantation to observe maternal toxicity and embryotoxicity, and find the sensitive exposure point of embryo implantation disorders induced by CS2. Animal treatment in part 2 was similar to part 1. Pregnant mice were sacrificed at different endpoints after exposed to CS2, and uterine tissues were collected to detect the protein and mRNA expression of mTOR, in order to analyze and explore the role of mTOR in the mechanism of embryo implantation disorder induced by CS2. Animal treatment in part 3 was similar to part 2. Part 3 detected protein and mRNA levels of integrin β3 and LIF in uterine tissues of pregnant mice to explore the mTOR, integrin β3, LIF and the relationship among them in the mechanism of embryo implantation disorder induced by CS2.Exposure points and endpoints:In this study, the exposure points of the three parts were the same. Depending on the exposure time, four groups were definite: exposure on GD3 (group 1); exposure on GD4 (group 2); exposure on GD5 (group 4); exposure on GD6 (group 4) and received single intraperitoneal injection of CS2 or solvents (631.4 mg/kg; solvent control for olive oil; injection capacity of O.lml/lOg weight). All of the endpoints in part 1 were GD9 for observing maternal toxicity and embryotoxicity, and recording the number of implanted embryos. There were 10 endpoints in par 2 and part 3. The number of examination endpoints in each group was different:Four endpoints in Group 1 were designed on GD4,5,6 and 7, respectively. Three endpoints in Group 2 were on GD5,6 and 7. Two endpoints in Group 3 were on GD6 and 7. One endpoint in Group 4 was on GD7. Consequently, there were 10 endpoints all together. Part 2 and part 3 were designed for collecting uterine tissue samples and testing the expression levels of mTOR, integrin P3 and LIF protein and mRNA.Animal treatment:After acclimating to the standard laboratory conditions for 1 week, male mice were allowed to mate with the females (1:1). The day of vaginal plug presented was designated as the first day of gestation (GD1). Gestational mice on GD1 were randomized into groups. Each endpoint consisted of twelve mice in which six had CS2 exposure and six olive oil exposure.Sample collection and detection of mTOR, integrin β3 and LIF:In part 1, mice on GD9 were sacrificed and gravid uteri were excised and weighted. The number of implanted embryos in each litter was counted; furthermore, litter weight of embryos was noted. The weights of uterus, ovary, liver, spleen and kidney on GD9 were recorded. Mice in part 2 and part 3 were sacrificed on the designated endpoints and gravid uteri were excised. Each uterine was cut into two parts and preserved at 80℃. Half of each uterine was used for protein analysis and the other for mRNA analysis. IHC (Immunohistochmeistry) and ELISA (enzymes linked immunosorbent assay) were used for detecting the expression of mTOR protein in uterine tissues. Real-time PCR was used for detecting the expression of mTOR mRNA in uterine tissues. BCA method was used for detecting the concentration of protein. SDS-PAGE and western blotting were used for detecting the expression of integrin β3 and LIF protein in uterine tissues.Statistical analysis:Results were analyzed by a computerized statistical program (SPSS 16.0).The experimental data was analyzed by a homogeneity test for variance, followed by a completely randomized design analysis of variance (ANOVA). If the variances show homogeneity, the different data among groups were determined by a Dunnett t test; if not, the data was analyzed by a nonparametric analysis of variance (Kruskal-Wallis H test and Mann-Whitney U test). Differences were considered statistically significant if P＜0.05. Values of the results are presented as means ± standard deviation.Results1. Maternal toxicity and embryotoxicityMaternal toxicity:No significant difference was observed in GD9 body weight, total body weight gain and net body weight gain among all the groups (P>0.05). There was also no statistically significant difference in absolute organ weights and relative organ weights-organ coefficient (adjusted with body weight) among these groupsEmbryotoxicity:In comparison with the control, the number of implanted embryos was clearly reduced in all CS2 exposed groups (P＜0.05). The rates of embryo implantation were decreased by 36.89%, 57.58%, 33.79% and 36.55% after CS2 exposure on GD3, GD4, GD5 and GD6, respectively, as compared to control. When compared to the controls, there were statistically significant changes in litter weight of embryos of all CS2 exposed groups, but no significant difference was observed among all of the groups in mean weight of embryos after adjustment to the number of implanted embryos.2. The expressions of mTOR proteins and mRNA in uterine tissueThe mTOR expression in GD4 exposure significantly declined at the endpoints of GD5 in comparison with the controls and the decreased rates was 56.42% (P<0.01). The expression of mTOR protein at the different endpoints of exposure group was lower than the control. The GD5 endpoint of GD4 exposure group has the lowest expression of mTOR. (P＜0.01) and GD7 endpoint of exposure group decreased by 45.22%(P＜0.01). Similarly, the expression of mTOR mRNA was lowest at the GD3 endpoint of GD5 exposure group, which decreased by 62% compared with the control (P＜0.01). The expression of mTOR of the first endpoint in different groups all decreased.3. The expressions of integrin β3 and LIF proteins and mRNA in uterine tissueAfter CS2 exposure, the expressions of integrin β3 and LIF of the first endpoint in different groups were all decreased (P＜0.05). And GD4 endpoint of GD5 exposure group was decreased by 29% and 54.3%, respectively, compared with the control (P＜0.05).All the expression of integrin β3 and LIF mRNA at endpoints of exposure groups decreased AND GD4 endpoint of GD5 exposure group was most markedly, which decreased by 85% and 45.9%, respectively (P＜0.01).Conclusions1. There was no obvious maternal toxicity but embryo toxicity after exposure to CS2 at the peri-implantation which meant the decreased number of implanted embryos. This phenomenon was most obvious in the GD4 exposure group, suggesting that GD4 was the most sensitive time to CS2 exposure.2. Exposure to CS2 at the peri-implantation could induce the decreased level of mTOR protein and mRNA and this phenomenon was most obvious at the first endpoint of the GD4 exposure group, which might contributed to embryo loss after CS2 exposure.3. Exposure to CS2 at the peri-implantation could induce the decreased protein and mRNA level of integrin β3 and LIF which was consistent with the change of mTOR, suggesting that three of them might be collaboratively involved in the mechanism of embryo implantation disorders induced by CS2, but the exact mechanism needs further study to verify. |
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